A decreased partial pressure of oxygen in the blood is detected in mammals by oxygen sensitive (type I) cells in the carotid body. Type I cells communicate this information through the carotid sinus nerve to the respiratory and cardiovascular networks in the brainstem which trigger adaptive responses. Dopamine, the primary neurotransmitter in type 1 cells, is released during hypoxia. Synthesis of dopamine is enhance during hypoxia by a mechanism involving increased activity of tyrosine hydroxylase (TH), the rate limiting enzyme in catecholamine synthesis. The investigators have recently shown that hypoxia stimulates TH gene expression in carotid body type I cells. Efforts to identify the molecular mechanisms that regulate TH gene expression during hypoxia have been hindered by the lack of an appropriate cell culture system. The investigators discovered recently that the clonal cell line derived from pheochromocytoma tumor (PC12 cells) can be used as an experimental model system for oxygen sensitive type I cells. The investigators showed that TH mRNA is increased in these cells in response to hypoxia by a mechanism that involves increases in both the rate of TH gene transcription and increased stability of TH mRNA. The major objective of the present study is to characterize the mechanism that regulates increased stability of TH mRNA during hypoxia in PC12 cells. Increased mRNA stability provides a means for adaptive enhancement of TH gene expression and therefore TH enzyme during hypoxia. The initial studies are designed to characterize the time course and magnitude of TH mRNA stability at different ranges of oxygen tensions in PC12 cells and in PC12 cells differentiated towards sympathetic-like neurons in response to nerve growth factor. A major mechanism for enhancing mRNA stability is the interaction of a protein binding factor to a region of the TH mRNA. Thus the next series of experiment are undertaken to attempt to identify the sequences on TH mRNA which bind regulatory proteins and determine if these sequences are able to confer increased mRNA stability. In another series of experiments, RNA mobility shift assays will be used to determine if redox potential or phosphorylation of regulatory proteins affect their ability to bind to TH mRNA. The preliminary experiments indicated that a heme containing protein may be involved in the regulation of TH mRNA stability during hypoxia. Inhibition of heme synthesis inhibits enhancement of TH mRNA stability by hypoxia. In addition, substitution of nickel or cobalt for iron in heme protein, which prevents binding of oxygen and locks heme in the deoxygenated state, mediates an increase in TH mRNA stability. Experiments are performed to further characterize the potential role of a heme protein in mediating TH mRNA stability during hypoxia.